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Cell Surface Targeting

Cell Surface Targeting. Two routes. Protein-protein interface. DNA-DNA interface. Protein-protein. Standard part. 5’ GTTTCTCC GAATTC GCGGCCGCT TCTAGA G EcoRI XbaI. T ACTAGT AGCGGCCG CTGCAG GGAGAAAC 3’ SpeI PstI. Protein domain.

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Cell Surface Targeting

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  1. Cell Surface Targeting

  2. Two routes Protein-protein interface DNA-DNA interface

  3. Protein-protein

  4. Standard part 5’ GTTTCTCCGAATTCGCGGCCGCTTCTAGAG EcoRIXbaI TACTAGTAGCGGCCGCTGCAGGGAGAAAC 3’ SpeI PstI Protein domain 5’ GTTTCTCCGAATTCGCGGCCGCTTCTAGA EcoRIXbaI ACTAGTAGCGGCCGCTGCAGGGAGAAAC 3’ SpeI PstI Domain assembly 5’ GTTTCTCCGAATTCGCGGCCGCTTCTAGA EcoRIXbaI ACTAGA ThrArg Mixed ACTAGTAGCGGCCGCTGCAGGGAGAAAC 3’ SpeI PstI Protein domain BioBricks

  5. Streptavidin • Found in bacteria Streptomyces avidinii • Full-length ~160 aa’s, core ~ 140 aa’s • Binds strongly to biotin (vitamin H or B7) • Kd ~ 10-15 M (Chaiet, 1964) • No cysteines, no carbohydrates, no charge McDevitt, 1999

  6. Lpp-OmpA surface expression • Lipoprotein (Lpp): major outer membrane protein (most abundant protein in E. coli by numbers); targeted to outer membrane • Outer membrane protein A (OmpA): well-characterized eight-strand beta-barrel transmembrane domain; stable surface xpression • Lpp 20aa signal peptide + Lpp aa 1-9 + OmpA aa 46-159 + surface protein • OmpA 46-66 also successful • Bla, Fv fragments, OPH, Cex (C. fimi) • Goal: Use Lpp-OmpA to express streptavidin on the surface to bind biotinylated DNA or protein to the membrane Francisco et al., 1992

  7. Progress/agenda • Created BioBricks of three streptavidin clones from Ting lab: wild-type, wild-type + His6 tag, dead mutant • Created BioBricks of full Lpp and OmpA, then Lpp(1-29), OmpA(46-66), OmpA(46-159) • More BioBricks: single-chain dimer streptavidin from Aslan lab • Assembly and expression of Lpp-OmpA-streptavidin

  8. DNA-DNA

  9. Aptamers • Short DNA/RNA sequences that have high specificity and affinity for substrate • Low generation time

  10. Pros • Potential control of binding kinetics and thermodynamics • Control of relative amounts of different substrates that bind cell surface • Swappable • Cons • Interaction strength limited by strength of aptamer-protein interactions

  11. First designs

  12. Progress • Have begun to characterize DNA-DNA interaction • Gel shift assays have failed to prove that aptamers are binding the proteins • For the future: more promising assays.

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